Dose response of powder sourced compounds for small molecule regulators of Bcl-2 family protein interactions, panel upload with wildtype and mutant Bfl1 and wildtype BclB.

One arm of apoptosis is regulated by the balance of anti-apoptotic and pro-apoptotic Bcl-2 family members. In humans, six genes have been identified that encode anti-apoptotic proteins characterized by the presence of conserved motifs designated as three Bcl-2 homology (BH) regions, BH1, BH2, and BH3 [Reed, et al. 2004]. These domains form a hydrophobic cleft in tertiary structure. Pro-apoptotic more ..

One arm of apoptosis is regulated by the balance of anti-apoptotic and pro-apoptotic Bcl-2 family members. In humans, six genes have been identified that encode anti-apoptotic proteins characterized by the presence of conserved motifs designated as three Bcl-2 homology (BH) regions, BH1, BH2, and BH3 [Reed, et al. 2004]. These domains form a hydrophobic cleft in tertiary structure. Pro-apoptotic family members contain BH3 regions that form an amphipathic helix, and these helices bind in the clefts of the anti-apoptotic proteins. Overexpression of pro-apoptotic BH3 peptides has been shown to increase apoptosis of leukemia cells using in vitro and animal model studies [Holinger, et al. 1999; Wang et al. 2000; Walensky et al. 2004]. The binding of fluorochrome-conjugated BH3 peptides (including Bim) to Bcl-2 family members thus provides the basis for construction of fluorescence-based assays amenable to flow cytometry high throughput screening for small molecule regulators of these interactions. The screen is a multiplexed assay to identify small molecule regulators of protein interactions between the BH3 peptide of Bim and the following six Bcl-2 family members: Bcl-XL, Bcl-W, Bcl-B, Bfl-1, and Mcl-1 and Bcl-2 (the eponymous founding member of the Bcl-2 family).

23 compounds were found active against Bfl-1 in AID 2080, a confirmatory dose response. Subsequent fluorescence polarization assay results declared all of these 23 compounds to be inactive. Two possibilities for this discrepancy were tested. One possibility was that a cysteine residue in the binding pocket of Bfl-1 was reacting covalently with the compounds, so a mutant was made in which the cysteine was replaced by an alanine at amino acid 55, denoted Bfl-1CA. The second possibility was that the original preparation of Bfl-1 had degraded during the year in a -80 degreeC freezer between the original screen (AID 1008) and the first confirmatory dose response assay (AID 2080). The present confirmatory dose response assay revealed that none of the compounds in AID 2080 were active using the fresh Bfl-1 or the fresh Bfl-1CA. We therefore declare that the results of AID 2080 are invalid, probably due to deterioration of the original preparation of Bfl-1.

The multiplex is constructed by using beads for each protein target that have been labeled with varying intensities of red color, so that each assay is built on a unique bead set, and each bead set is associated with a unique optical address. Beads are first washed in HPSMTB buffer for 20 minutes before adding the appropriate GST-Bcl fusion protein. The bead sets (ThermoFisher Scientific product numbers XPR-1687-XPR-1696), have similar size (~ 4 micron diameter) and are distinguished by distinct emission characteristics at 665 +/-10 nm with excitation at 635 nm. Thus, GST-Bfl-1 might be non-covalently coated onto red level 1 beads, GST-Bcl-B onto red level 2 beads, etc. The bead sets (each with bound protein) and uncoated beads (see below) are first centrifuged separately, then combined and centrifuged again, and finally diluted just before loading into 384-well plates, to minimize bead-protein dissociation before the assay begins.

The HTS assay was conducted in 384-well microplates in a total assay volume per well of 10.1 microliters (5 microliters of bead mixture, 0.1 microliters of test compound, and 5 microliters of 100 nM F-Bim in HPSMTB). Test compound concentration was 10 microM. Controls, which contained bead mixture and F-Bim but no test compound, were located in columns 1 and 2 on each plate. Plates were placed horizontal axis on rotators and incubated for 1-2 hours at 4 degrees C.

A glutathione-only bead set control (no associated GST-protein) was incorporated into each well as a fluorescence scavenger to determine inherent fluorescent properties (at 530 nm emission) of the test compounds. Specificity of F-Bim binding was determined with a Positive Control using a block of the F-Bim fluor with a non-fluoresceinated Bim peptide. The F-Bim blocking control was run daily as a separate single tube assay using Bim at 5 microM.

The primary screen yielded hit compounds that were evaluated in previous dose response screens. In the study reported here, a series of compounds were re-ordered in powder form and a confirmational dose response evaluation was done. Test compounds at 10 mM concentration in DMSO were serially diluted 1:3.16 eight times for a total of nine different test compound concentrations. Final compound dilutions in DMSO ranged from 1 microM to 10 mM. These dilutions were then diluted 1 to 100 to give an assay concentration range of 10 nanoM to 100 microM. The compounds were tested in either duplicate or triplicate dose response curves.

Sample acquisition and preliminary analysis is conducted with the HyperCyt(R) high throughput flow cytometry platform. The HyperCyt system interfaces a flow cytometer and autosampler for high-throughput microliter-volume sampling from 384-well microtiter plates [Kuckuck, et al. 2001]. The stream of particles is excited at 488 nm and 635 nM, and flow cytometric data of light scatter and fluorescence emission at 530 +/- 20 nm (FL1) and emission at 665 +/- 10 nm (FL8) are collected on a Cyan Flow Cytometer (Dako). Analysis of the time-resolved acquisition data file uses IDLeQuery software to merge the flow cytometry data files with compound worklist files generated by HyperSip software. The raw data are parsed in IDLeQuery to produce annotated fluorescence summary data for each well. The parsed data are then processed through an Excel template file constructed specifically for the assay to segregate data for each target and the fluorescence scavenger in the multiplex. Gating based on forward scatter (FS) and side scatter (SS) parameters is used to identify singlet bead populations. Gating based on FL8 emission distinguishes the beads coated with different proteins, and the green median fluorescence intensity (MFI) per bead population (well) is calculated.

Calculations:

In dose response experiments, the assay was performed without compound and with nine different concentrations of compound, from 10 nanoM to 100 microM, to produce a series of 9 data points. HyperView calculates the median channel fluorescence (MCF) for each of these ligand concentrations, generating competition curves. The percent inhibition response was calculated by the following equation:% response = 100 x (DMSO - SampleFL)/(DMSO -BlockCntrl)where all variables are Median Fluorescence Intensity associated with the bead set bound with a specific protein. SampleFL is for beads in wells containing test compound, DMSO is the plate average of wells without test compounds, and BlockCntrl is for measurement in presence 5 micromolar non-fluorescent Bim. Baseline of % response is 0%.

These % response values were fitted by Prism(R) software (GraphPad Software, Inc., San Diego, CA) using nonlinear least-squares regression in a sigmoidal dose response model with variable slope, also known as the four parameter logistic equation. Curve fit statistics were used to determine the concentration of added test compound competitor that inhibited fluorescent ligand binding by 50 percent (EC50, microM), the low and high boundaries of the 95% confidence interval of the EC50 estimate, the Hill Slope, and the correlation coefficient (r squared) indicative of goodness-of-fit. Note that an compound eliciting inhibitory responses would have positive Hill Slope and a compound eliciting activating responses would have negative Hill Slope due to the data being fitted are the % inhibition response.

Fluorescence of the compounds could be green, which would increase the apparent amount of F-Bim bound (signal). None of such artifacts occurred with the large dynamic range of this assay, and no effort was made to annotate them. Fluorescence of the compounds could be red, which would shift the bead sets out of their gates, which had been set using whole plate data. Again, none of such artifacts occurred (bead numbers decreased at the upper end of a few dose response experiments), and no effort was made to annotate them.

Compounds with EC50 less than 10 microM and magnitude of response greater than 40% (i.e., Bottom of sigmoidal curve < 0.6 * Top of sigmoidal curve, listed as FIT_PERCENT_SPAN) were said to be "Active", and thus given a PUBCHEM_ACTIVITY_SCORE. The PUBCHEM_ACTIVITY_SCORE for each individual panel was based on the following equation;